Method for controlling absorption of cholesterol

ABSTRACT

THE METHOD OF ORALLY ADMINISTERING COMPOSITIONS FOR CONTROLLING ABSOPRTION OF CHOLESTEROL ARE DESCRIBED HEREIN, THESE COMPOSITIONS CONTAINING AN EFFECTIVE DOSE OF A HEPTAENE POLYENIC MACROLIDE SELECTED FROM THE CLASS CONSISTING OF CANDICIDIN, FUNGIMYCIN, AMPHOTERICIN B, TRICHOMYCIN, HAMYCIN, CANDIDIN AND AYFACTIN.

United States Patent Office 3,714,348 Patented Jan. 30, 1973 3,714,348 METHOD FOR CONTROLLING ABSORPTION OF CHOLESTEROL Harry W. Gordon, Bronx, N.Y., and Carl P. Schaffner,

Trenton, N.J., assignors to Julius Schmid Inc., New

York, N.Y.

No Drawing. Continuation of application Ser. No. 627,313, Mar. 31, 1967, now Patent No. 3,627,879, dated Dec. 14, 1971. Divided and this application Dec. 2, 1970, Ser. No. 94,593

The portion of the term of the patent subsequent to June 8, 1988, has been disclaimed Int. Cl. A61k 21/00 U.S. Cl. 424117 8 Claims ABSTRACT OF THE DISCLOSURE The method of orally administering compositions for controlling absorption of cholesterol are described herein, these compositions containing an effective dose of a heptaene polyenic macrolide selected from the class consisting of candicidin, fungimycin, amphotericin B, trichomycin, hamycin, candidin and ayfactin.

This application is a continuation of application \Ser. No. 627,313 filed Mar. 31, 1967, now U.S. Pat. No. 3,627,879.

This invention relates to the method of controlling cholesterol absorption in mammals. I

The implication of the blood lipids as a contributing factor in the functioning of the highly vascularized organs such as the kidneys, liver, brain, lungs, testes, heart, and other organs requiring smooth, free, sufficient blood flow has contributed to the intensive and vigorous research for agents, that will control the lipid content, including triglycerides, cholesterol, lipoproteins, etc., in the bloodstream and tissues. A majority of studies have focused on cholesterol because of the substantial evidence available relating blood cholesterol levels to conditions such as atherosclerosis, arteriosclerosis and other sclerotic conditions, coronary heart disease, cerebral hemorrhage, liver and kidney dysfunction associated with vascular obstruction, sterol calculi resulting from hypercholesterolemia, etc.

Diseases such as arteriosclerosis, which is a generic term for a number of chronic pathologic conditions, affect the intima or the media of arteries and is characterized by thickening, hardening and loss of elasticity of vessel walls, with resultant alteration in size of the lumen. Atherosclerosis, which is a form of arteriosclerosis is characterized by intimal thickening due to localized accumulations of lipids, known as atheromas. Atherosclerosis is of great importance because of its predilection for coronary (cerebral) and peripheral arteries. It develops insidiously, probably due to multiple factors (metabolic, humoral and hemodynamic being of primary significance).

The earliest lesions of atherosclerosis are the subintimal fatty streaks seen in the thoracic aorta in young mammals, which either retrogress or grow larger, forming plaques. While these may involve any artery, they are most common in the aorta, coronary, cerebral, and peripheral arteries of the lower extremities. Subintimal hemorrhage or ulceration of the plaque may lead to thrombosis and occlusion of the involved vessel, resulting in a variety of symptoms and signs due to ischemia.

It is believed that B-lipoproteins are basically responsible for the disease process. These lipoproteins are a heterogeneous family of macromolecules containing protein, cholesterol, phospholipids and triglycerides in varying proportions. The chemical measurement of .any of these lipids is an index of the accumulation of lipoproteins of a given density. These lipoproteins, which are essential but not solely sufficient to cause disease, interact with the arterial wall in such a way as to produce the discrete intimal lesion.

The most frequent and important cause of acute renal failures (dysfunction) is acute tubular necrosis (lower nephron nephrosis or kidney shutdown). Although the causes advanced for this condition are numerous it is postulated that lipoid material depositing on the capillary walls may also contribute to the condition. Functional renal disorders are also associated with arteriolar nephrosclerosis which consists of sclerosis of the small renal arterioles. There is also interference with the nor mal function of the kidney in cases of atherosclerosis in which there is a thickening of the peripheral arteries due to localized accumulations of lipids known as atheromas.

Liver dysfunction associated wtih vascular obstruction is often seen in obese mammals in whom it is due to excessive fat intake. Fatty infiltration of the liver can also be caused by numerous factors including chronic infections (e.g., tuberculosis), metabolic disorders (e.g., diabetes mellitus), etc. In these conditions, fatty liver probably is due to migration of fat from storage deposits.

There has also been in recent years increasing interest in the formulation of fat-free diets in order to control obesity and/or the amount of lipids, including cholesterol, present in the bloodstream as it is well known that the ingestion of fats is one means of increasing the amount of lipids in the blood. There has been much publicity of the fact that the ingestion of fat, which essentially consists of glycerol esters of higher fatty acids which break down in the digestive tract, must be maintained at a minimum in cases where a high lipid metabolism is considered dangerous.

Despite intensive research for hypolipoidal agents, iii-- eluding hypocholesterolemic agents, relatively few compounds have been found which are acceptable for longterm use and even these have drawbacks. For example, natural and synthetic estrogens are known to inhibit cholesterol induced atherosclerosis in mammals. In general, however, the undesirable feminizing side effects of estrogens limit their usefulness in male mammals. These side effects have stimulated a search for non-estrogenic estrogens (compounds in which the sterolic effect has been separated form. the estrogenic effect) but to date no useful agent has been discovered. Other hypocholesterolemic agents have met with varying degrees of success but undesirable side effects have been encountered. In addition, the use of a fat-free diet to alter the lipoidal metabolism has obvious limitations because fat is used extensively in the preparation of many foods and hence such a diet is extremely difficult to maintain.

It has now been unexpectedly discovered that the oral administration in a solid pharmaceutical formulation of a polyenic macrolide compound will be effective in reducing and/or controlling triglycerides, cholesterol, lipoproteins, etc. in mammals, thereby providing an agent useful for the treatment of those conditions believed associated with the lipid metabolism, e.g., hypocholesterolemia and other conditions mentioned heretofore. This discovery is particularly surprising because it has been suggested in the prior art that Mycostatin (trademark for nystatin), a polyenic macrolide compound having four conjugated double bonds, is ineffective on the serum cholesterol (Steiner et al., Circulation, vol. 24, pp. 729-735 (1961)).

.The action of the compounds of this invention is believed not due to the antibiotic function of the polyenic macrolide compounds which have been previously described in the art, but apparently to their chemical structure.

Accordingly, one aspect of the present invention is to provide a method for reducing and/or controlling cholesterol in mammals which comprises orally administering an effective dose of a polyenic macrolide compound.

Another aspect of the present invention is to provide an orally administered composition for inhibiting absorption of cholesterol from the intestinal tract which composition comprises a solid pharmaceutical formulation containing an effective dose of a polyenic macrolide compound.

An additional aspect of the present invention is to provide an enteric tablet or capsule containing an effective dose of a composition of the present invention.

Other aspects of the invention will be apparent from the following detailed description.

As used herein, the reduction of sterol levels is intended to include the treatment of hypersterolemia, e.g., hypercholesterolemia, as well as conditions believed associated directly or indirectly with hypersterolemia.

The known polyenic macrolide compounds have been produced as antibiotics by cultivation of Streptomyces in different media and by extraction of the substances from these cultures. It has been demonstrated in the literature that the known polyenic compounds are (1) of fairly high molecular weight (ca. 700-15), (2) contain macrocylic lactones, better known as macrolides (hereinafter referred to as polyenic macrolide compounds), and (3) each possess a chromophore in the nucleus of from four to seven conjugated double bonds (tetraenes, pentaenes, hexaenes, and heptaenes) identified by examination of their ultra-violet absorption spectra. These conjugated systems are generally unsubstituted (except the methyl pentaenes) and either of the all-trans or cis-trans configuration. Based on the evidence available to date, it is indicated that the known polyenic macrolide compounds .contain a twenty-six to a thirty-seven membered lactone ring wherein all of the ring atoms except the single oxygen atom are carbons. The evidence to date also indicates that only C, H, O, and N are present in-the known polyenic macrolide compounds.

The following articles may be consulted for references to the discovery, isolation and chemical properties of the heptaene polyenic macrolides:

('1) Vining, The Polyene Antifungal Antibiotics, Hindustan Antibiotics BulL, vol. 3, pp. 32-54 (1960).

2) Waksman et at, The Acti-nomycetes, vol. III, Antibiotics of Actinomycetes (Williams and Wilkins, Baltimore, 1962).

(3) Droughet, Noveaux Antibiotiques Antifongiques," Symp. Int. Chimiotherapie, Naples, 1961, pp. 21-50 1963 (4) W. Oroshnik et al., Fortschritte der Chemie Organischer Naturstoffe, vol. XXI, pp. 18-79 (1963).

Although the specific polyenes named supra, are identified and described in the literature, references are given here for abundance of identification to various patents and well known publications. Thus, candicidin is described in US. Pat. No. 2,992,162 issued July 1, 1961; Physicians Desk Reference (19th edition, 1964); and the Merck Index of Chemicals and Drugs (7th edition).

Amphotericin B is described in US. Pat. 2,908,611 issued Oct. 13, 1959; and Merck Index of Chemicals and Drugs (7th edition); Martindales Extra Pharmacopedia (25th edition).

Fungimycin is described in US. Pat. 3,182,004, issued May 4, 1965 and the Merck Index of Chemicals and Drugs 8th ed.).

Hamycin is described in US. Pat. 3,216,751 issued July 19, 1966 and in Martindales Extra Pharmacopedia (25th ed.) published February 1967.

Candidin is described in the Merck Index of Chemicals and Drugs (7th edition).

Trichomycin is described in Martindales Extra Pharmacopedia (25th edition); and the Merck Index of Chemicals and Drugs (7th edition).

Ayfactin is described in British Pat. No. 796,982 issued in 1958 and in Antibiotics & Chemotherapy, vol. 8, pp. 491-495 (1958). Cultures for producing this material are availaable under the following identificaiton numbers: NRRL 2209, NRRLB 1286, NRRLB 1287, NRRLB 1288 and ATCC 11989.

It will be understood that where a polyenic macrolide compound described in the art is identical with one of the above specifically named polyenic macrolide compounds, but has been known by another name by reason of independent production or production in accompaniment to other antibiotics, the identification of such substances by the name set forth above is intended to mean the same compound under all other designations.

The pharmaceutical compositions are formulated so as to be suitable for oral administration. The active ingredient is contained in a capsule or tablet, preferably in enteric form. The quantity of effective dose supplied by each capsule or tablet is relatively unimportant since the total dosage can be reached by administration of either one or a plurality of capsules or tablets or both. The capsules employed may comprise any well known pharmaceutically acceptable material such as gelatin, cellulose derivatives, etc. The tablets may be formulated in accordance with conventional procedure employing solid carriers, lubricants, etc., well known in the art. Examples of solid carriers are: starch, sugar, bentonite and other commonly used carriers.

The following examples illustrate suitable pharmaceutical formulations containing the compounds of this invention.

EXAMPLE 1 Hard gelatin capsule available from the Robin Pharmacal Corporation .(size 00) is filled with about 0.83 gram of lactose (Fast Flow available from Foremost Dairies, Inc.) and about mg. of active material, the lactose and active ingredient being triturated together in a pestle and mortar until a very fine yellow amorphous powder resulted, prior to filling of the capsule. Obviously, any desired number of capsules may be filled by mixing together any amount of lactose and active ingredient in the same weight ratio indicated above so that each capsule will contain 100 mg. active ingredient; and the quantity of active ingredient may be altered, as desired by varying the weight ratio of the indicated materials.

EXAMPLE 2 g. of corn starch and 2112.5 g. lactose are dried at F. for 12 hours before compounding. After drying, each of these materials is sifted through a No. 14 mesh stainless steel screen. The sifted corn starch and lactose are thoroughly mixed for 30 minutes and to this mixture there is added a blended mixture of 250 g. active ingredient and 12.5 g. magnesium stearate. This admixture is blended and then compressed on a tableting machine into 5000 substantially round tablets each containing 50 mg. active ingredient and weighting about 500 mg.

EXAMPLE 3 Enteric tablets for use in this invention. may be formulated as follows:

16 g. of powdered corn starch (U .S.P. quality) is dried at 120 F. for 12 hours and passed through a N0. 25 mesh stainless steel screen. The sifted corn starch is then mixed with 255 g. of anhydrous lactose (direct tablet grade). To this mixture, 4 g. of magnesium stearate is added followed by 50 g. of the active ingredient. These materials are then mixed in a small pebble mill for 30 minutes and compressed on a single punch machine producing 1,000 tablets, each containing 50 mg. active ingredient. Each tablet weighs approximately 325 mg. The average hardness is 6, as measured on a Monsanto Hardness Tester.

The tablets are then placed in a coating pan rotating at 29 r.p.m. and subjected to warm air of approximately 80 F. for about 10 minutes. Then'30 cc. of a pharmaceutical glazed composition is applied, this composition being refined wax and rosin-free orange flake shellac with anhydrous alcohol as the medium therefor. Talcum (U.S.P.) or similar dusting powder is applied to the tablets to prevent the tablets from sticking to each other or to the pan and this procedure is followed after the application of each coat to the tablets. The coat is allowed to dry for approximately one hour. Thereafter three additional coats are applied in a similar manner, each coat comprising 30 cc. of the pharmaceutical glaze, with approximately one hour of drying time between the application of successive coats. After four coats are applied the tablets are dried overnight at room temperature and then four more coats are applied in the same manner using the same composition. Each coat is allowed to air dry for 3 hours before applying the next coat. Each of the 8 coats of the enteric tablets is approximately 0.001 inch in thickness. Obviously, the thickness of the coating can be controlled by varying the concentration of the pharmaceutical glaze in the alcohol medium.

The enteric tablets are tested in accordance with the in vitro disintegration test for enteric-coated tablets described in U.S.P. XVII and were found to pass this test.

While the number of coats used in the example heretofore described is 8, it will be appreciated that there are many factors to be considered which permit variation in the number of coats, including the size and shape of the tablets or capsules, the type of coat or combination of coats, etc.

Other procedures and materials well known in the prior art may be employed to prepare suitable enteric coatings. The selection of the coating substance is governed to a large extent by pH and enzyme considerations and the desire to have the enteric composition disintegrate or dissolve when it reaches the duodenum region of the intestinal tract and not in the stomach. The disintegration or dissolution of an enteric coating in the intestinal tract usually depends on several factors, the most important of which are (1) the presence of acidic groups in the enteric substance which cause it to be insoluble in the low pH environment of the stomach but soluble in the intestinal tract due to the higher (but usually not alkali) pH of the media there, and (2) the resistance of the coating to attack by oral and gastric enzymes.

Illustrative of other well known substances that may be used for the enteric coating are the following: cellulose acetate phthalate with resinous carrier; cellulose are stabilized on diet, feeding regime. After the average control serum lipid values are obtained, 20 mg./kg. of body weight is orally administered twice daily, once in the morning and the second dose about 6 to 8 hours later. After two weeks of oral administration, blood samples are tested for serum cholesterol. All blood samples are drawn for assay prior to feeding with no food (except water) for at least 12 hours. Administration of candicidin is continued and blood samples are tested again for serum cholesterol at the end of each week of administration.

While the present invention is not predicated on any present theoretical considerations, it is believed that the possible mechanism by which the compositions of this invention exhibit their action is through the formation of a complex in the intestinal tract with sterols, such as cholesterol, thus preventing absorption of the complexed sterol. Therefore, absorption of the compositions of this invention is not necessary for alteration of the lipid metabolism to occur. Initially, depleting the absorption of the sterols is likely to stimulate a release of stored materials from the tissues (fatty acids, triglycerides, sterols, etc.) which in some instances may result in an initial increase in serum levels which will then be followed by a decrease after equilibration is reached. Preventing absorption of cholesterol has numerous applications including treatment of hypercholesterolemia and conditions associacetate phthalate-tolu balsam-shellac; cellulose acetate phthalate with fats and waxes; shellac-Castor oil; ammoniated shellac; shellac-stearic acid-tolu balsam; stearic acidcastor oil over shellac-silica gel, cellulose acetate phthalates with or without plasticizer and dusting powder(s); acid phthalates of glucose, fructose, etc.; ternary copolymers of styrene, methacrylic acid and butyl half-ester of maleic acid; alkyl resin-unsaturated fatty acids-shellac; polyvinyl acid phthalate, etc.

For a description of the procedure for manufacturing enteric formulations such as those exemplified heretofore, reference should be made to U.S. Pats. Nos. 2,196,768; 2,433,244; 2,455,790; 2,540,979; 2,858,252; 3,080,346 and British Pats. Nos. 760,403 and 820,495.

The effectiveness of the compounds of this invention has been indicated by tests in large mammals, i.e., those weighing at least abount 1 kilogram. For example, tests conducted on dogs with candicidin demonstrates the effectiveness of the polyenic macrolide compounds including the reduction of blood cholesterol levels.

The basic procedure used for the determination of cholesterol levels is the method of J. P. Peters and D. D. Van Slyke, described in the text Quantitive Clinical Chem., vol. II, pp. 504-508 (Williams & Wilkins) and the fatty acid content was also determined in accordance with the procedure described at page 478 of that text.

Average control serum lipid levels are established prior to the administration of the candicidin to the dogs which ated therewith and in dietary control as discussed at pages 1-4 of the specification.

It is preferred, commensurate with the desideratum of obtaining the highest degree of efiectiveness of the compositions of this invention per given dose of active ingredient, to use an enteric tablet or capsule. Thus when using a specific known polyene macrolide compound in the form of an enteric solid, the entire compound will remain intact when it reaches the intestinal tract so long as the enteric coating composition retains its integrity in the stomach. On the other hand, administration of the same dose in a standard solid pharmaceutical formulation may result in a cleavage of any amino sugar present, or of other groups similarly sensitive to gastric conditions. Such cleavage may further result in alteration of the polyenic marcolide nucleus, thereby diminishing the effetciveness of the active ingredient.

The effective dosage of the compounds of this invention depends upon the severity of conditions, the stage and the individual characteristics of each mammal being treated. It is expected that the compositions will generally be administered in a dosage range from about 1 mg. to about mg. active ingredients per kg. of body weight per day and preferably from about 5 mg. to about 40 mg. per kg. of body weight per day.

The compositions of this invention may in addition contain dietary supplements such as vitamins, choline, salts of glycerophosphoric acid and inositol, which are known to be effective in reducing serum cholesterol levels.

What is claimed is:

1. A process of inhibiting the absorption of cholesterol from the intestinal tract of a mammal in need of such treatment which comprises orally administering to said mammal an effective dose for inhibiting the absorption of cholesterol of a composition containing a polyenic macrolide selected from the group consisting of candicidin, fungimycin, amphotericin B, tirchomycin, hamycin, candidin and ayfactin.

2. A process according to claim 1 wherein said effective dose comprises about 1 mg. to about 40 mg. per kilogram of body weight per day of said polyenic macrolide.

3. A process according to claim 2 wherein said effective dose is administered in a solid form selected from the group consisting of a capsule and a tablet.

4. A process according to claim 2 wherein said polyenic macrolide is administered in enteric coated form.

5. A process according to claim 2 wherein said polyenic macrolide is candicidin.

6. A process according to claim 2 wherein said polyenic macrolide is amphotericin B.

7. A process according to claim 2 wherein said polyenic macrolide is fungimycin.

8. A process of inhibiting the absorption of cholesterol from the intestinal tract of a mammal in need of such treatment which comprises orally administering to said mammal about 1 mg. to about 40 mg. per kilogram of body weight per day of candicidin in enteric coated form.

References Cited Louria: Antibiotics and Chemotherapy, -vol. 5, No. 5, pp. 295-301 (1958).

JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. 

